The present invention relates to diagnostic assays, specifically an assay that detects a modified form of AT-III as a predictor of thrombotic and pre-thrombotic condition.
Antithrombin (AT-III), the primary plasma inhibitor of blood coagulation enzymes, is a member of a large superfamily of structurally related proteins which includes several serine proteinase inhibitors (serpins). These proteinase inhibitors share a common mechanism by which a covalent, essentially irreversible complex is formed with the target enzymes. During this process, the serpin is cleaved by the protease at the reactive center, allowing the inhibitor to change from its normal strained conformation to a relaxed conformation and resulting in the inactivation of the inhibitor. Several studies have shown that proteases can cleave serpins near their reactive center in such a way that the inhibitor is inactivated, but the protease is not.
It is possible to perform an evaluation of this sort of inactivation in vitro using purified components, but determination of its relevance in vivo where a large excess of active inhibitor exists is more difficult. Another possible approach would be to generate monoclonal antibodies specific for the cleaved form of the inhibitor and use them in an ELISA format. Although this method can work well, it is time consuming and relatively expensive.
Interestingly, several of these serine protease inhibitors are themselves the target of attack by proteinases of mammalian and non-mammalian origin. In the case of AT-III a specific and inactivating cleavage is known in vitro to be catalyzed by human neutrophil elastase (HNE) in a heparin-dependent fashion near the active site of the sequence of the inhibitor without any apparent effect in HNE activity. HNE is secreted by neutrophils during inflammation and may affect functional AT-III levels in vivo. This may contribute to the development of life-threatening thrombosis accompanying widespread activation of neutrophils in inflammation and septicemia.
Maintenance of plasma concentrations of AT-III at or near its normal level of approximately 2 micromolar is apparently essential to avoid a tendency toward blood clotting in both hereditary deficiency and disease. Disease related decreases, which can be more than 50% in extreme cases of septicemia, have often been attributed to the consumption of AT-III in the course of inhibiting coagulation enzymes.
The recently described in vitro inactivation of AT-III by neutrophil elastase (Jochum et al., Z. Physiol. Chem. 362:103, 1981) suggests a possible alternative explanation. Nevertheless, the extrapolation of this observation to the physiologic setting must take into account the very high plasma levels of a specific elastase inhibitor. This inhibitor, alpha-1 proteinase inhibitor, normally prevents expression of elastolytic activity in plasma.
The treatment of acquired antithrombin (AT-III) deficiencies, such as disseminated intravascular coagulation (DIC), septicemia and other inflammatory diseases, has been thwarted by the inability to diagnose the early onset of a pre-thrombotic state. Treatment of acquired deficiencies with AT-III or other elastase inhibitors has been delayed until clinical symptoms manifest themselves. Delayed treatment has had equivocal results. These equivocal results are thought to reflect the unpredictable severity and rate of onset of the disease state, as well as the likelihood of inadequate treatment doses.
Currently, determinations of functional plasma antithrombin constitute the only diagnostic test for ascertaining AT-III consumption or turnover during pathologic states. While these may give some information during or after major thrombotic episodes, they are insensitive to the prethrombotic condition. The consumption of AT-III in a limited or localized reactive zone, i.e., the formation of a clot on the vascular lining, would generally have little effect on the total plasma pool of antithrombin. Localized events within the vasculature, even when locally severe, have little impact on overall AT-III levels which are normally high (2 micromolar). The current clinical tests measuring AT-III levels are inadequate to diagnose potentially small changes occurring in pre-thrombotic conditions.
The anticoagulant heparin is a functionally heterogeneous sulfated carbohydrate with the ability to bind to AT-III and accelerate its rate of inhibition of coagulation enzymes. Heparin does not normally circulate in blood but, rather, is an integral component of vascular endothelial cell walls.
The anticoagulant activity of heparin, as purified from animal tissues and administered therapeutically, derives from a limited subfraction of heparin molecules (35%) possessing affinity for antithrombin.
Jochum et al., Eur. Surg. Res., 13:152-168 (1981) disclose that a protease inhibitor, aprotinin, may prevent the decline in plasma proteins in dogs made endotoxemic by infusion of E. coli endotoxin. Aprotinin may therefore be a candidate for treatment of DIC.
Jochum et al., Hoppe-Seyler's Z. Physiol. Chem. Bd., 362, S.: 103-112 (1981) disclose that granulocytic (neutrophil) elastase cleaves AT-III in vitro to produce a modified AT-III, and that some of the AT-III consumption in septicemia or endotoxemia may be due to proteolysis of AT-III.
Carrell et al., Nature, 317:730-732 (1985) further describe the conformational change brought about by elastase cleavage of AT-III.
Jochum et al., Adv. Exp. Med. Biol., 167:391-404 (1984) describe a study of AT-III, alpha-1-PI and other protein levels in septic patients. They found a significant decrease of AT-III activity in septic patients, presumably as a result of AT-III depletion of elastase released by neutrophils as part of the inflammatory response.
Biologically active AT-III has been expressed in E. coli transfected with human cDNA, as disclosed in Bock et al. U.S. Pat. No. 4,632,981.
Herion et al., Blood, 65(5):1201-7 (1985) describe monoclonal antibodies to AT-III.
Lau et al., J. Biol. Chem., 254(18):8751-8761 (1979) disclose making and using polyclonal antibodies directed to identified fragments of prothrombin.
Other immunoassays for AT-III are described in the literature. Fujisawa et al. describe a latex agglutination assay used to measure AT-III levels in healthy persons and patients with DIC; the results were comparable with a radial immunodiffusion assay (Chem. Abstr., 94:13481, 1981).
E. McKay, Brit. J. Hemat., 46:277-285 (1980), describes an electroimmunoassay for AT-III. One antiserum used differentiated active AT-III from AT-III-protease complexes. Anomalies incurred with previous AT-III assays are described.
Bick et al , Throm. Res., 10:721-729 (1977) describe an assay for AT-III which was used to monitor AT-III levels in DIC patients.
However, none of these prior art assays measure elastase modified (inactivated) AT-III (ATx). The measurement of AT-III is insensitive with regard to potential thrombotic states in that normal AT-III levels are much higher than needed for normal physiological coagulation balance. A substantial imbalance resulting in significant generation of ATx would only result in a slight (e.g. 5%) decrease in AT-III levels.
Normal individuals, however, possess no ATx. Detection of ATx suggests a pathological state. In addition, measurement of AT-III-protease complexes is hindered by the short circulation half-life of these complexes, on the order of 10 minutes. ATx, however, has a half life in the order of that of AT-III.
Detecting and measuring circulating elastase modified antithrombin (ATx), based on this mechanism, provides for early diagnosis and hence, treatment of the pre-thrombotic and thrombotic conditions. Thus, there remains a need for a reliable, efficient method of detecting ATx in serum samples.